Linaclotide is a GC-C (enterocyte uridylate cyclase C) receptor agonist and was approved by US FDA in August 2012 for the treatment of adult chronic idiopathic constipation and constipation-predominant irritable bowel syndrome (IBS-C). Linaclotide, which is developed by Ironwood Pharmaceuticals, is a polypeptide consisting of 14 amino acids and containing three disulfide bonds in chemical structure thereof, and can be prepared by cell expression and chemical synthesis. Linaclotide has a sequence of Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr (SEQ ID NO: 1) and a structure of:

SEQ ID NO: 1 is being submitted as an ASCII text file via EFS-Web, file name Sequence-1, size 461 bytes, created on Jun. 13, 2018, the content of which is incorporated herein by reference.
Benitez et al. published a relevant report on Peptide Science in 2010. Three methods are adopted in the report to try to synthesize linaclotide. In method (1), Trt is used to protect the side chain of Cys, and the linear peptide is synthesized using an Fmoc solid phase peptide synthesis process to obtain a crude peptide, which then is oxidized by one-step using a liquid-phase oxidation process to obtain the target peptide. In method (2), Trt and Acm are used to protect the side chain of Cys, and a partially protected linear peptide is obtained by an Fmoc solid phase peptide synthesis process, and then the synthesis of disulfide bonds is completed using a semi-selective strategy. In method (3), three completely selective strategies are used to synthesize linaclotide, respectively: [2 Mmt+2 Acm+2 Trt], [2 Acm+2 Trt+2 pMeOBzl] and [2 StBu+2 Trt+2 pMeOBzl].
In method (1), the synthesis steps are simple, and only one kind of protecting group for Cys is used. However, for peptides where three disulfide bonds need to be formed site-specifically, many different disulfide-bond mismatched isomers will be obtained by random oxidation. Although it is possible to make the conversion into target molecules in the oxidation process as high as possible by some buffer solution systems, the production of other isomers cannot always be avoided. Such a procedure easily results in a crude target peptide with a lower purity and yield, making it very difficult to achieve large-scale production. Meanwhile, oxidation by this method has a very high dependence on external conditions such as temperature, etc. Under different circumstances, the product yield obtained by natural oxidation also varies greatly, which is not conducive to the control of product quality. Method (2) is a method for semi-selective site-specific oxidation, in which one disulfide bond is site-specifically oxidized, decreasing the number of different isomers formed, as compared with method (1). However, the production of isomers still cannot be avoided. Meanwhile, it is further mentioned directly in the report that two disulfide bonds are formed using iodine oxidation, and the yield of crude peptides is severely reduced. In method (3), the researchers chose to use three different complete selectivity methods to form three disulfide bonds, but no target product is obtained.
At present, there is still no method for preparing linaclotide with a high efficiency. Chinese patents CN 104231051A, CN 104628826A, CN 104163853A, CN 104844693A, and CN 102875655A introduce a method for forming three disulfide bonds by one-step oxidation, wherein a linaclotide resin is firstly synthesized, and then cleaved to remove all protecting groups and resin solid phase carrier to obtain a linaclotide linear crude peptide, which is finally subjected to a one-step oxidation reaction using an oxidation system. Among them, a GHS/GSSH oxidation system is used in CN 104231051A, CN 104163853A, and CN 102875655A; elemental iodine is used to oxidize in a sodium phosphate buffer solution of pH=6˜13 in CN 104628826A; and a cysteine hydrochloride/DMSO buffer solution oxidation system is used in CN 104844693A. Although the one-step oxidation method can convert the linear peptide into the target structure as much as possible by the buffer system, the disulfide bond mismatched isomer impurities still cannot be avoided to produce and the yield is lower.
Therefore, it is necessary to explore a linaclotide synthesis method which is performed under mild conditions, low in cost, high in yield, high in product purity, simple and stable in processes, and suitable for large-scale production.